Downregulation of TRPC6 regulates ERK1/2 to prevent sublytic C5b‑9 complement complex‑induced podocyte injury through activating autophagy.

Abstract

Idiopathic membranous nephropathy (IMN) is a common glomerular disease, in which 50-60% of patients can progress to end-stage renal disease within 10-20 years, seriously endangering human health. Podocyte injury is the direct cause of IMN. Sublytic C5b-9 complement complex induces damage in podocytes' structure and function. In sublytic C5b-9 treated podocytes, the expression of canonical transient receptor potential 6 (TRPC6) is increased. However, the specific mechanism of TRPC6 in sublytic C5b-9 treated podocytes is unclear. The present study aimed to reveal the effect and mechanism of TRPC6 on sublytic C5b-9-induced podocytes. Normal human serum was stimulated using zymosan to form C5b-9. A lactate dehydrogenase release assay was used to examine C5b-9 cytotoxicity in podocytes. The RNA and protein expression levels were analyzed using reverse transcription-quantitative PCR, western blotting and immunofluorescent assay, respectively. Cell Counting Kit-8 assay and flow cytometry were carried out to test the viability and apoptosis of podocytes, respectively. Transmission electron microscopy was used to observe autophagic vacuole. F-actin was tested through phalloidin staining. Sublytic C5b-9 was deposited and TRPC6 expression was boosted in podocytes stimulated through zymosan activation serum. Knockdown of TRPC6 raised the viability and reduced the apoptosis rate of sublytic C5b-9-induced podocytes. Meanwhile, transfection of small-interfering (si)TRPC6 facilitated autophagy progression and enhanced the activation of cathepsin B/L in sublytic C5b-9-induced podocytes. The phosphorylation level of ERK1/2 was receded in siTRPC6 and sublytic C5b-9 co-treated podocytes. Moreover, the addition of the ERK1/2 activator partially reversed the effect of TRPC6 inhibition on sublytic C5b-9-induced podocytes. TRPC6 knockdown reduced the damage of sublytic C5b-9 to podocytes by weakening the ERK1/2 phosphorylation level to activate autophagy. These results indicated that targeting TRPC6 reduced the injury of sublytic C5b-9 on podocytes.